DESCRIPTION (applicant's abstract): Mast cells are located perivascularly close to nerve processes and can secrete many vasoactive and proinflammatory molecules. In addition to allergic triggers, mast cells can also be activated by direct nerve stimulation, by neuropeptides, and by acute immobilization stress through the local release of corticotropin releasing hormone (CRH) or urocortin (Ucn), which is more potent than CRH. Intradermal injections of CRH or Ucn induced rat skin mast cell activation and increased vascular permeability, both of which were inhibited by pretreatment with neutralizing antiserum to CRH or the CRH-receptor antagonist antalarmin. CRH or acute stress-induced skin vascular permeability measured with Evans blue extravasation was absent in W/W (v) mast cell deficient mice, but was present in their wt controls indicating it is mast cell dependent; this finding was supported by the fact that vascular permeability was also blocked by the "mast cell stabilizer" disodium cromoglycate (cromolyn). Similar results were obtained in rats and mice in response to acute immobilization stress. The in vivo, but not in situ, CRH action was absent in rodents treated with capsaicin to deplete sensory nerve fibers of their substance P (SP) content and was also inhibited by the neurotensin (NT) receptor antagonist SR48692. We are hypothesizing that acute stress releases CRH and/or Ucn in the skin leading, directly or through neuropeptides such as SP and NT, to mast cell activation and increased vascular permeability; leukocyte infiltration may then contribute to local inflammation and possibly to delayed-hypersensitivity (DTH) reactions. We propose to use normal and genetically deficient mice to investigate: (1) the effect of acute stress on (a) skin mast cell activation determined by image analysis, as well as residual skin histamine and mouse mast cell protease (MMCP)-6 content in CRH knock-out mice, and (b) vascular permeability quantitated by 99Technicium-gluceptate (99Tc) extravasation in CRH knock-out and W/W (v) mice and their +/+ controls; (2) any change in skin dorsal root ganglia (DRG) or spinal cord CRH, Ucn or CRH receptor expression after stress, using immunohistochemistry, Western and Northern analysis or RT-PCR; (3) the involvement of SP on stress-induced mast cell activation and vascular permeability in SP and NK-1 receptor knock-out mice, as well as the possible sequence of action of CRH or SP using combination of knock-out animals and CRH or SP-receptor antagonists; (4) the effect of CRH and Ucn on secretion of histamine, IL-6 and MMCP-6 or tryptase, respectively, from mouse purified skin and human umbilical cord-derived mast cells stimulated immunologically or by SP. These studies will help us understand how acute stress triggers skin mast cell activation, increased vascular permeability and possibly DTH. They will also help investigate the pathophysiology of skin syndromes exacerbated by stress, such as atopic dermatitis or psoriasis, especially since CRH and CRH receptors have been identified in human skin.